The long term objectives of this study are to develop a model of the ion transporting systems in the placental syncytiotrophoblast and to investigate the involvement of ion transporters in the common pathophysiologies of pregnancy. The initial section of this project was concerned with identifying and characterizing the major ion transporters in the syncytiotrophoblast using microvillous and basal membrane vesicles prepared from normal, term placenta. This research has advanced to the point that a change in the experimental approach is now necessary. The characterization of an individual transporter in membrane vesicle establishes the potential activity of the transporter but does not provide information on its integration into the cellular ion transport system, nor determine the contribution it makes to specific cellular functions, and does not describe the regulatory mechanisms operative in vivo. It is essential therefore to investigate the function of these transporters in a cellular system. The recently developed syncytiotrophoblast culture provides the opportunity to study ion transport systems in the cellular context. A second and important aspect of the experimental approach to this study is the use of high resolution, fluorescence microscopy, employing ion-sensitive probes to measure intracellular ion concentrations. The advent of this technique and the synthesis of new ion-specific fluorescent probes provides the ability to examine rapid rates of change in intracellular ion concentrations, repetitively and over extended time periods. The specific aims of the project during the next period are (1) to continue investigation and characterization of individual transporters using vesicles, looking particularly for those transporters which are activated or regulated by nucleotide binding or phosphorylation; (2) to determine the intracellular pH and intracellular concentrations of sodium, potassium and chloride in resting syncytiotrophoblast; (3) to examine the operation of ion transport systems which maintain ionic homeostasis, provide for transcellular (transplacental) transport, regulate intracellular pH and control cellular volume in the syncytiotrophoblast; (4) to measure the effects of hypoxia on the time course of intracellular ion concentrations in cultured syncytial cells, determining the reversibility, effects on ion-coupled substrate transport and the effectiveness of interventions designed to prolong cellular viability; (5) to develop a new model for the investigation of ion transport, the superfused, chorionic villous fragment which, when loaded with ion-specific probes, will provide information on ion transport systems from primary cells retaining their in vivo orientation and connections to other villous tissue elements. The ion transport systems to be investigated are the essential core underlying a range of placental functions and knowledge of these systems is crucial to our understanding of maternal-fetal interaction. In addition, for the first time an investigation will be carried out, exploring the reaction of placental ion transport systems to a commonly encountered problem of pregnancy, fetal hypoxia.